Biologic Scaffold For Prevention of Pulmonary Fibrosis

ABSTRACT

Provided herein are methods of preventing, lessening or treating pulmonary fibrosis in a subject. The methods comprise delivering an amount of a powdered extracellular matrix (ECM)-derived material to the respiratory system of the subject effective to prevent, lessen or treat pulmonary fibrosis in a subject. Also provided is an apparatus for delivering the powdered ECM-derived material to a subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.13/132,708, which is a national stage of International PatentApplication No. PCT/US2009/066754, filed Dec. 4, 2009, which claims thebenefit of U.S. Provisional Patent Application No. 61/200,949, filedDec. 5, 2008, each of which is incorporated herein by reference in itsentirety.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with government support under Grant No. R01HL63700, awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

Provided herein are compositions and related methods useful in promotingnormal remodeling and preventing fibrosis in the lung.

Fibrosis is the formation or development of excess fibrous connectivetissue in an organ or tissue as a reparative or reactive process, asopposed to a formation of fibrous tissue as a normal constituent of anorgan or tissue. Pulmonary fibrosis is a fibrosis (fibrotic condition)involving the lung. Pulmonary fibrosis may arise from damage to the lungtissue, as is present in interstitial lung disease (ILD, also known asdiffuse parenchymal lung disease). ILD may be classified according tothe cause, for example and without limitation: inhaled substances,including silicosis, asbestosis, berylliosis and hypersensitivitypneumonitis; drug induced, such as from antibiotics, chemotherapeuticdrugs (e.g., bleomycin) and antiarrhythmic agents; connective tissuedisease, such as systemic sclerosis, dermatomyositis, systemic lupuserythematosus and rheumatoid arthritis; infection, such as atypicalpneumonia, pneumocystis pneumonia (PCP) and tuberculosis; idiopathic,such as sarcoidosis, idiopathic pulmonary fibrosis and Hamman-Richsyndrome; or malignancy, such as lymphangitic carcinomatosis.

Idiopathic pulmonary fibrosis (IPF) is a debilitating diseasecharacterized by inflammation, fibroblast proliferation, and excessiveextracellular matrix deposition in the lung. The pathogenesis ofpulmonary fibrosis has been studied widely in animal models. The mostwidely used model to study pulmonary fibrosis in rodents is thebleomycin model (Meltzer E B, et al. Idiopathic pulmonary fibrosis.Orphanet J Rare Dis 2008; 3:8 and Moeller A, et al. The bleomycin animalmodel: A useful tool to investigate treatment options for idiopathicpulmonary fibrosis? Int J Biochem Cell Biol 2008;40(3):362-382). Whilethe mechanisms for injury are still not fully understood, it appearsthat the pathology begins with epithelial damage followed by activationof alveolar macrophages and infiltration of the lung tissue bycirculating inflammatory cells that release cytokines such as tumornecrosis factor (TNF)-α and interleukin (IL)-1β. Ultimately, thispro-inflammatory environment leads to the recruitment and proliferationof fibroblasts/myofibroblasts that produce transforming growth factor(TGF)-β and deposit large quantities of collagenous, fibrotic tissue.Currently, pulmonary transplantation is the only viable option forpatients with IPF (Meltzer E B, et al. Idiopathic pulmonary fibrosis.Orphanet J Rare Dis 2008; 3:8 and Moeller A, et al. The bleomycin animalmodel: A useful tool to investigate treatment options for idiopathicpulmonary fibrosis? Int J Biochem Cell Biol 2008; 40(3):362-382).

SUMMARY

Provided herein are compositions, drug products, apparatus and methodsfor prevention and/or treatment of pulmonary fibrosis. It has been foundthat delivery of ECM-derived materials, for example powdered orsolubilized ECM-derived materials, to a subject's respiratory systemlessens development of fibrosis and improves outcome in subjects exposedto conditions that result in pulmonary fibrosis, as is the case with,for example, ILD.

The ECM-derived material may be isolated and prepared from (derivedfrom), for example and without limitation, urinary bladder tissue,trachea or lung tissue. In one non-limiting embodiment, theextracellular matrix-derived material comprises epithelial basementmembrane and, optionally, subjacent tunica propria. In another, theextracellular matrix-derived material comprises tunica submucosa. In yetanother, extracellular matrix-derived material comprises epithelialbasement membrane, subjacent tunica propria and tunica submucosa. Incertain non-limiting embodiments, the extracellular matrix-derivedmaterial is isolated from small intestinal submucosa or the dermis ofthe skin.

Methods of preventing or lessening development of pulmonary fibrosis ina subject also are provided. The methods comprise, without limitation,delivering to an airway of a subject an ECM-derived material to reduce,lessen or prevent (e.g., an amount effective to reduce, lessen orprevent) development of fibrosis in a subject exposed to a pulmonaryfibrosis-causing agent or condition. The ECM-derived material may bepowdered or solubilized. The ECM-derived material may be administeredprior to or after exposure to a fibrosis-inducing event, such asexposure to a chemotherapeutic agent, radiotherapy, asbestos, etc. Thepulmonary fibrosis-causing agent or condition (e.g., event) includes,without limitation, agents that cause ILD, including, withoutlimitation, the following conditions: silicosis, asbestosis, berylliosisand hypersensitivity pneumonitis; drug induced, such as fromantibiotics, chemotherapeutic drugs (e.g., bleomycin) and antiarrhythmicagents; radiation-induced fibrosis, often arising from radiotherapy, forexample in cancer patients; exposure to a chemical agent, such asmustard gas; bronchiolitis obliterans; connective tissue disease, suchas systemic sclerosis, dermatomyositis, systemic lupus erythematosus andrheumatoid arthritis; infection, such as atypical pneumonia,pneumocystis pneumonia (PCP) and tuberculosis; idiopathic, such assarcoidosis, idiopathic pulmonary fibrosis and Hamman-Rich syndrome; ormalignancy, such as lymphangitic carcinomatosis.

An apparatus is provided according to certain embodiments of the presentinvention. The apparatus comprises an airway delivery system comprisinga composition comprising an ECM-derived material, such as a powderedand/or solubilized ECM-derived material. In certain embodiments, theairway delivery system is a metered dose inhaler, a device which is ableto deliver to a patient one or more unit doses of a dry powder oraerosolized (e.g., nebulized or spray) liquid composition. Thecomposition may be a dry powder or a liquid that can be aerosolized. Thecomposition, e.g. in a metered dose inhaler, may comprise a propellantto assist in aerosolizing and propelling the drug product into asubject's respiratory system. The powdered ECM-derived material may haveany effective size range, mean and distribution that can be effectivelydelivered to the respiratory system, and particularly the lungs of asubject to prevent or lessen development, or treat pulmonary fibrosis.In one example, the powdered ECM-derived material has a maximum particlesize of 250 μM (meaning the ECM-derived material can pass through ascreen having a mesh size of 250 μM); a maximum particle size of 75 μM;or a maximum particle size of 5 μM. In one non-limiting embodiment, theECM-derived material comprises basement membrane and tunica propriaobtained from porcine urinary bladder. In another embodiment, theECM-derived material is digested, solubilized ECM.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a cross-sectional view of the wall of theurinary bladder (not drawn to scale). The following structures areshown: epithelial cell layer (A), basement membrane (B), tunica propria(C), muscularis mucosa (D), tunica submucosa (E), tunica muscularisexterna (F), tunica serosa (G), tunica mucosa (H), and the lumen of thebladder (L).

FIG. 2 is a graph showing that UBM-ECM attenuates bleomycin-inducedpulmonary fibrosis. Fibrosis was measured in wild type C57BL/6 mice 14days after exposure. Average histology score was determined by a blindedpathologist and the scoring system was as follows: 0=no fibrosis,1=0-25% fibrosis, 2=25-50%, 3=50-75%, 4=75-100%. **p<0.001.

FIG. 3. UBM-ECM prevents bleomycin-induced fibrosis. Wild-type mice wereeuthanized 14 days after intratracheal administration of bleomycin.Histological analyses revealed that UBM-ECM limited pulmonary fibrosis:A. Vehicle B. Bleomycin (BLM) C. BLM & UBM-ECM D. BLM & SIS-ECM E. BLM &UBM-ECM Digest F. BLM & Pepsin Digest E. Average histology score wasdetermined by a pathologist (T.D.O.) blinded to treatment groups. Thescoring system was as follows: 0=no fibrosis, 1=0-25% fibrosis, 2=25-50%fibrosis, 3=50-75% fibrosis, 4=75-100% fibrosis. All scores werereported as the percentage of fibrosis when compared to bleomycintreatment alone (100% fibrosis) except UBM-ECM digest, which wasnormalized to its control pepsin digest. Vehicle image is representativeof the following treatments: saline, UBM-ECM, SIS-ECM, UBM-ECM digest,or pepsin digest. Results shown are representative of n=4-7/treatmentgroup. *p<0.05 when compared to control, †p<0.05 when compared tobleomycin, ¥p<0.05 when compared to bleomycin+pepsin digest, one-wayANOVA.

FIG. 4. Treatment with ECM does not prevent bleomycin-induced leukocyteaccumulation. All bleomycin-treated mice have increased total cells intheir BALF (A-C) and specifically significantly more macrophages andmodest increases in neutrophils and lymphocytes (D-F) regardless of ECMtreatment. Results shown are representative of three independentexperiments (n=4-7/group/experiment). Vehicle=saline or TiO2 controls.*p<0.05 compared to vehicle control, †p<0.05 compared to UBM-ECMcontrol, ¥p<0.01 compared to vehicle and UBM-ECM control, §p<0.05compared to BLM or BLM+Pepsin Digest

FIG. 5. ECM treatment does not affect bleomycin toxicity. A549 cells(5,000 cells/well) were serum starved for 4 hrs and then incubated withserum-free F12K media, 0.02 units of bleomycin (BLM), and BLM withindicated amounts of UBM-ECM (A), SIS-ECM (B), or UBM-ECM digest (C) for24 hours. Cell viability was measured using Promega CellTiter 96®AQueous Non-Radioactive Cell Proliferation Assay according tomanufacturer's instructions (OD₄₉₀) and reported as the percentage ofviable cells when compared to media control. *p<0.05 when compared tomedia control.

FIG. 6. ECM promotes chemotaxis of epithelial cells. Chemotaxis ofserum-starved A549 cells (30,000 cells/well) was quantitativelyevaluated utilizing the Neuro Probe 48-well microchemotaxis chamber.Chemotaxis toward F12K media with 10% FBS (positive control), serum-freeF12K media, and 100 μg/mL and 500 μg/mL UBM-ECM digest or control pepsindigest was measured in quadruplicate in three independent experiments.The average percentage of migrated cells for each condition wasnormalized to the average percentage of cells that migrated toward themedia with 10% FBS (positive control) in each experiment. *p<0.05 whencompared to 10% FBS

FIG. 7. UBM-ECM stimulates re-epithelialization. A549 cell monolayerswere serum-starved for 24 hrs, wounded, and exposed to UBM-ECM digest,bleomycin (BLM), BLM with UBM-ECM powder or digest, or BLM with pepsindigest control. Results were reported as the percentage of change inwound width over 24 hrs. *p<0.05 when compared to UBM-ECM digest controltreatment

FIG. 8 is a graph showing the effects of ECM on development of pulmonaryfibrosis from treatment with a chemotherapeutic agent, as described inExample 3

DETAILED DESCRIPTION

The use of numerical values in the various ranges specified in thisapplication, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges are both preceded by the word “about”. In this manner,slight variations above and below the stated ranges can be used toachieve substantially the same results as values within the ranges.Also, unless indicated otherwise, the disclosure of these ranges isintended as a continuous range including every value between the minimumand maximum values. For definitions provided herein, those definitionsrefer to word forms, cognates and grammatical variants of those words orphrases.

Described herein are methods for preventing, lessening or treatingpulmonary fibrosis in a subject, as well as drug products useful in suchmethods. The methods include delivery of an extracellular matrix-derived(ECM-derived) material to the respiratory system, including the nasalcavity, trachea and lungs of a subject in an amount effective toprevent, lessen or treat pulmonary fibrosis in a subject.

Biologic scaffolds composed of mammalian extracellular matrix (ECM) havebeen shown to promote site-specific remodeling of musculoskeletal,cardiovascular, urogenital, and dermal tissues. The species and tissueorigin of the ECM scaffolds vary, but the most commonly used and studiedECM scaffolds are derived from porcine small intestinal submucosa (SIS)and urinary bladder matrix (UBM). The mechanisms by which these ECMscaffolds promote tissue remodeling are also not fully understood, butappear to include the presentation of a three-dimensionalmicroenvironment supportive of cell growth and migration that transmitsbiochemical and mechanical cues to the cells, and rapid degradation withsubsequent release of small peptide fragments that possess innatebioactivity, specifically chemotaxis for progenitor cells andantibacterial behavior. This combination of microenvironmental cues andrelease of matricryptic peptides is responsible for the presence of anaccommodative, tissue remodeling response as opposed to a cytotoxic,pro-inflammatory immune response. ECM scaffolds that are fullydecellularized and not chemically crosslinked elicit a predominantly M2type macrophage response.

ECM scaffolds have recently received attention for treatment of airwayinjury in several pre-clinical models. Recent studies have shown thatECM scaffold materials can prevent air leakage into the pleural cavitywhen used as a primary treatment or when used as reinforcement for asurgical staple line after partial lung resection (Downey D M, et al.Functional comparison of staple line reinforcements in lung resection.Ann Thorac Surg 2006; 82(5):1880-1883; Downey D M, et al. Functionalassessment of a new staple line reinforcement in lung resection. J SurgRes 2006; 131(1):49-52; and Gilbert T W, et al. Repair of the thoracicwall with an extracellular matrix scaffold in a canine model. J Surg Res2008; 147(1):61-67). SIS-ECM used for reinforcement of surgical staplelines showed significant improvement in intrabronchial pressures ascompared to other reinforcement materials. Repair of the lung with aUBM-ECM scaffold showed moderately dense well-organized collagenoustissue formation at the site of resection without evidence ofinflammation, necrosis, or scarring in the lung. In addition, UBM-ECMhas recently been shown to promote the formation of a pseudostratified,columnar, ciliated epithelium when used for patch tracheoplasty in acanine model (Gilbert T W, et al. Morphologic assessment ofextracellular matrix scaffolds for patch tracheoplasty in a caninemodel. Ann Thorac Surg 2008; 86(3):967-973; discussion 973-974).

ECM-derived material can be used for a large number of medicalapplications including, but not limited to, wound healing, tissueremodeling, and tissue regeneration. For example and without limitation,the scaffold can be used for wound healing. As used herein a compositionmanufactured from an ECM-derived material is useful in preventing andtreating pulmonary fibrosis. The ECM-derived material is shown toameliorate or lessen the occurrence of fibrosis in instances wherepulmonary fibrosis can develop or progress. As an example, pulmonaryfibrosis may develop as part of the progress of ILD, which, as indicatedabove, can have a variety of causes, including and without limitation:inhaled substances, including silicosis, asbestosis, berylliosis andhypersensitivity pneumonitis; drug induced, such as from antibiotics,chemotherapeutic drugs (e.g., bleomycin) and antiarrhythmic agents;connective tissue disease, such as systemic sclerosis, dermatomyositis,systemic lupus erythematosus and rheumatoid arthritis; infection, suchas atypical pneumonia, pneumocystis pneumonia (PCP) and tuberculosis;idiopathic, such as sarcoidosis, idiopathic pulmonary fibrosis andHamman-Rich syndrome; or malignancy, such as lymphangiticcarcinomatosis.

A “drug product” is a compound, composition, formulation, apparatus,etc. comprising an “active agent”, which is a compound or compositionthat has a specific pharmacological effect. A drug product is preferable“pharmaceutically acceptable,” meaning that it is suitable foradministration to a subject for a stated purpose, which is typicallyreferred to as an “indication”. Irrespective of whether a drug productor other composition, compound or material may or may not cause harm byits administration to a subject, it may be “pharmaceutically acceptable”if benefits of the composition, compound, material or drug productoutweigh its risks. In one aspect, a compound, composition, material,excipient or drug product is pharmaceutically acceptable if it meets therequirements of an applicable regulatory agency, such as the US Food andDrug Administration, or any other applicable regulatory body, whichincludes compounds, compositions, materials or drug products that areGenerally Recognized As Safe (GRAS). In a broader sense, a composition,compound, material or drug product and it is material is notbiologically or otherwise undesirable for its intended use, that is, thematerial can be administered to an individual along with the relevantactive compound without causing clinically unacceptable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the pharmaceutical composition in which it is contained.

Throughout the description and claims of this specification the word“comprise” and other forms of the word, such as “comprising” and“comprises,” means including but not limited to, and is not intended toexclude, for example, other additives, components, integers, or steps.

As used in the description and the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a composition”includes mixtures of two or more such compositions.

As used throughout, by a “subject” is meant an animal or human. Thus,the “subject” can include domesticated animals, such as cats, dogs,etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.),laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.) andbirds. In one aspect, the subject is a mammal such as a primate or ahuman, including patients.

As used herein, the term “polymer” refers to both synthetic polymericcomponents and biological polymeric components. “Biological polymer(s)”are polymers that can be obtained from biological sources, such as,without limitation, mammalian or vertebrate tissue, as in the case ofcertain extracellular matrix-derived (ECM-derived) compositions.Biological polymers can be modified by additional processing steps.Polymer(s), in general include, for example and without limitation,mono-polymer(s), copolymer(s), polymeric blend(s), block polymer(s),block copolymer(s), cross-linked polymer(s), non-cross-linkedpolymer(s), linear-, branched-, comb-, star-, and/or dendrite-shapedpolymer(s).

The ECM-derived material is preferably biocompatible. By“biocompatible,” it is meant that a polymer compositions and its normalin vivo degradation products are substantially non-toxic andnon-carcinogenic in a patient within useful, practical and/or acceptabletolerances. In one non-limiting embodiment, the compositions, and/ordevices are “biocompatible” to the extent they are acceptable for use ina human veterinary patient according to applicable regulatory standardsin a given jurisdiction. In another example the biocompatible material,when administered to a patient, does not cause a substantial adversereaction or substantial harm to cells and tissues in the body, forinstance, the polymer composition or device does not cause necrosis oran infection resulting in harm to tissues.

As used herein, the terms “extracellular matrix” and “ECM” refer to acomplex mixture of structural and functional biomolecules and/orbiomacromolecules including, but not limited to, structural proteins,specialized proteins, proteoglycans, glycosaminoglycans, and growthfactors that surround and support cells within mammalian tissues.

Generally, any type of extracellular matrix (ECM) can be used to preparethe ECM-derived material (for example and without limitation, see U.S.Pat. Nos. 4,902,508; 4,956,178; 5,281,422; 5,352,463; 5,372,821;5,554,389; 5,573,784; 5,645,860; 5,771,969; 5,753,267; 5,762,966;5,866,414; 6,099,567; 6,485,723; 6,576,265; 6,579,538; 6,696,270;6,783,776; 6,793,939; 6,849,273; 6,852,339; 6,861,074; 6,887,495;6,890,562; 6,890,563; 6,890,564; and 6,893,666; each of which isincorporated by reference in its entirety). By “ECM-derived material” itis meant a composition that is prepared from a natural ECM or from an invitro source wherein the ECM is produced by cultured cells and comprisesone or more polymeric components (constituents) of native ECM.

According to one non-limiting example of the ECM-derived material, ECMis isolated from a vertebrate animal, for example, from a warm bloodedmammalian vertebrate animal including, but not limited to, human,monkey, pig, cow, sheep, etc. The ECM may be derived from any organ ortissue, including without limitation, urinary bladder, intestine,trachea, lung, liver, heart, esophagus, spleen, stomach and dermis. TheECM can comprise any portion or tissue obtained from an organ,including, for example and without limitation, submucosa, epithelialbasement membrane, tunica propria, etc. In one non-limiting embodiment,the ECM is isolated from urinary bladder, which may or may not includethe basement membrane. In another non-limiting embodiment, the ECMincludes at least a portion of the basement membrane. The ECM materialmay or may not retain some of the cellular elements that comprised theoriginal tissue such as capillary endothelial cells or fibrocytes.

In one non-limiting embodiment, the ECM is harvested from urinarybladders (also known as urinary bladder matrix or UBM), for example andwithout limitation, porcine urinary bladders. Briefly, the ECM can beprepared by removing the urinary bladder tissue from a pig and trimmingresidual external connective tissues, including adipose tissue. Allresidual urine is removed by repeated washes with tap water. The tissueis delaminated by first soaking the tissue in a deepithelializingsolution, for example and without limitation, hypertonic saline (e.g.1.0 N saline), for periods of time ranging from ten minutes to fourhours. Exposure to hypertonic saline solution removes the epithelialcells from the underlying basement membrane. Optionally, a calciumchelating agent may be added to the saline solution. The tissueremaining after the initial delamination procedure includes theepithelial basement membrane and tissue layers abluminal to theepithelial basement membrane. This tissue is next subjected to furthertreatment to remove most of the abluminal tissues but maintain theepithelial basement membrane and the tunica propria. The outer serosal,adventitial, tunica muscularis mucosa, tunica submucosa and most of themuscularis mucosa are removed from the remaining deepithelialized tissueby mechanical abrasion or by a combination of enzymatic treatment (e.g.,using trypsin or collagenase) followed by hydration, and abrasion.Mechanical removal of these tissues is accomplished by removal ofmesenteric tissues with, for example and without limitation, Adson-Brownforceps and Metzenbaum scissors and wiping away the tunica muscularisand tunica submucosa using a longitudinal wiping motion with a scalpelhandle or other rigid object wrapped in moistened gauze. Automatedrobotic procedures involving cutting blades, lasers and other methods oftissue separation are also contemplated. After these tissues areremoved, the resulting ECM consists mainly of epithelial basementmembrane and subjacent tunica propria (layers B and C of FIG. 1).

In another embodiment, the ECM is prepared by abrading porcine bladdertissue to remove the outer layers including both the tunica serosa andthe tunica muscularis (layers G and F in FIG. 1) using a longitudinalwiping motion with a scalpel handle and moistened gauze. Followingeversion of the tissue segment, the luminal portion of the tunica mucosa(layer H in FIG. 1) is delaminated from the underlying tissue using thesame wiping motion. Care is taken to prevent perforation of thesubmucosa (layer E of FIG. 1). After these tissues are removed, theresulting ECM consists mainly of the tunica submucosa (layer E of FIG.1).

The ECM can be sterilized by any of a number of standard methods withoutloss of function. For example and without limitation, the material canbe sterilized by propylene oxide or ethylene oxide treatment, gammairradiation treatment (0.05 to 4 mRad), gas plasma sterilization,peracetic acid sterilization, or electron beam treatment. Treatment withglutaraldehyde results in sterilization as well as increasedcross-linking of the ECM. This treatment substantially alters thematerial such that it is slowly resorbed or not resorbed at all andincites a different type of host remodeling, which more closelyresembles scar tissue formation or encapsulation rather thanconstructive remodeling, which may not be desirable in the context ofthe present invention. If desired, cross-linking of the protein materialwithin the ECM can also be induced with, for example and withoutlimitation, carbodiimide isocyanate treatments, dehydrothermal methods,and photooxidation methods. In one non-limiting embodiment, the ECM isdisinfected by immersion in 0.1% (v/v) peracetic acid, 4% (v/v) ethanol,and 96% (v/v) sterile water for two hours. The ECM material is thenwashed twice for 15 minutes with PBS (pH=7.4) and twice for 15 minuteswith deionized water. The ECM-derived material may be further processedby optionally drying, desiccation, lyophilization, freeze drying,glassification, etc. The ECM-derived material optionally can be furtherdigested, for example and without limitation by hydration (if dried),acidification, enzymatic digests with, for example and withoutlimitation, trypsin or pepsin and neutralization, producing, forexample, a solubilized ECM-derived material having increased solubilityin a solvent, such as a water-based solvent.

Commercially available ECM preparations can also be used to manufacturethe ECM-derived material described herein. In one non-limitingembodiment, the ECM is derived from small intestinal submucosa or SIS.Commercially available preparations include, but are not limited to,Surgisis™, Surgisis-ES™, Stratasis™, and Stratasis-ES™ (Cook UrologicalInc.; Indianapolis, Ind.) and GraftPatch™ (Organogenesis Inc.; CantonMass.). In another non-limiting embodiment, the ECM is derived fromdermis. Commercially available preparations include, but are not limitedto Pelvicol™ (sold as PermacoP in Europe; Bard, Covington, Ga.),Repliform™ (Microvasive; Boston, Mass.) and Alloderin (LifeCell;Branchburg, N.J.). In another embodiment, the ECM is derived fromurinary bladder. Commercially available preparations include, but arenot limited to UBM (Acell Corporation; Jessup, Md.).

Gilbert, T. W., et al. (Biomaterials, 2005. 26(12): p. 1431-5)) describethe preparation of one non-limiting embodiment of ECM-derived material.In that method, ECM is isolated from porcine urinary bladder. Thebladder is harvested immediately following euthanasia of market weightpigs (approximately 120 kg). The ECM isolation is performed by removingthe tunica muscularis externa and tunica submucosa layers, leaving thebasement membrane and tunica propria intact. The UBM is then washed in a0.1% peracetic acid solution for 2 h with subsequent rinses in phosphatebuffered saline and distilled water to disinfect the material and removeany cellular remnants. Two methods may be used to produce a particulateform of UBM. The first method involved lyophilizing the disinfectedmaterial and then chopping it into small sheets for immersion in liquidnitrogen. The snap frozen material is then reduced to small pieces witha blender so that the particles were small enough to be placed in arotary knife mill, such as a Wiley mill. A #60 screen can be used torestrict the collected powder size to less than 250 mm. A Sonic sifteror other classification device can be used to remove larger particlesand/or to obtain a particle size distribution within a desired range.

Another method described in Gilbert et al. is similar to the previousmethod except the disinfected material is first soaked in a 30% (w/v)NaCl solution for 5 min. The material is then snap frozen in liquidnitrogen to precipitate salt crystals, and lyophilized to removeresidual water. This material is then comminuted as described in above.By precipitating NaCl within the tissue, it is expected that theembedded salt crystals would cause the material to fracture into moreuniformly sized particles. The particles are then suspended in deionizedwater and centrifuged for 5 min at 1000 rpm three times to remove theNaCl. The suspension is snap frozen and lyophilized again. Finally, thepowder is placed in a rotary knife mill to disaggregate the individualparticles.

Sonic sifting and laser diffraction can be used to analyze the particlesize distribution that resulted from powdering methods, such as the twopowdering methods described above. Sonic sifting involves separating thepowder by size through a series of graduated screens stacked in avertical configuration. The powder passes through the screens as aresult of sonic pulses along the longitudinal axis of the stack andmechanical agitation in the plane of the screens. The screen sizes,according to one non-limiting embodiment, can be 212, 125, 90, 63, and38 mm. Finer screens (e.g., electroform meshes) are available, forscreening down to 5 μM or less using a Sonic Sifter.

A laser diffraction method may be used to determine the powder size.Each particle diffracts the light, and the diffraction angle isinversely proportional to the size of the particle. The diffractionpattern can be detected using an array of detectors, and the particlesize can be calculated based on the angle and intensity observed.

The powdered ECM-derived material can have a range of particulate sizes.By “powdered” or “powder”, it is meant that the material has aparticulate size distribution sufficiently small so that the materialcan be inhaled or otherwise delivered to the lungs in sufficientlocation and quantity to produce the desired and/or stated effect,namely lessening or prevention of pulmonary fibrosis. The powder istypically prepared as a composition in any suitable form for delivery toa subject's airway, for example as a dry powder or combined with aliquid and/or gas in any suitable composition. The optimal sizes anddistributions can be determined by routine experimentation, for example,by methods described in the Examples below. Useful particle sizes anddistributions include the ranges: less than 250 μM (micrometers, ormicrons), less than 200 μM, less than 175 μM, less than 150 μM, lessthan 125 μM, less than 100 μM, less than 75 μM, less than 50 μM, lessthan 25 μM, less than 10 μM or less than 5 μM, for example less than 75μM or less than 10 μM. Size distributions can vary greatly, with meanparticle sizes ranging from, for example and without limitation, 200 μM,175 μM, 150 μM, 125 μM, 100 μM, 75 μM, 50 μM, 25 μM, 10 μM or 5 μM,depending on the method of classifying (size fractioning) the powder.The powder can be prepared by any useful method, for instance byphysical comminution, crushing, grinding, milling, spray drying, etc.Size fractionation of the particles can be accomplished by any effectivemethod, such as by sieving, sifting, screening, sedimentation,elutriation, etc. See, e.g., Troy, D. B., Editor, Remington: The Scienceand Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins(2005), pp. 702-719 for further description of methods of manufacturingpowders.

In one embodiment, the ECM-derived material is solubilized, e.g., bydigestion with a protease, such as pepsin, trypsin, collagenase, etc.The solubilized ECM-derived material can then be suspended in any usefulpharmaceutically-acceptable solution for use in any airway deliverysystem, including sprayers, aerosolizers, nebulizers, etc., for exampleas described herein. Also, the solubilized ECM-derived material may bedried, spray-dried, lyophilized, etc. and comminuted as is necessary toprepare one non-limiting example of a suitable dry powder for eitherlong-term storage or delivery to a subject via a dry powder dosingmethod, e.g., as are described herein.

The composition can be delivered by any suitable airway delivery devicein any fashion, including, without limitation: spraying, aerosolizing,nebulizing, inhaling, or otherwise delivering the composition into avolume of air for dispensation into a subject's airway. Airway deliverydevices may contain and deliver only a single dose of a composition suchas a drug product, but typically carry multiple (more than one) doses ofthe composition or drug product, and are referred to as meter doseinhalers. As part of the apparatus described herein, a large number ofdevices are commercially available for dosing and deliveringcomposition(s) in both dry or liquid (or otherwise) form to a subject'sairway. “Airway” is intended to include all parts of a subject'srespiratory system extending from the nose and mouth to the lungs, andincluding, without limitation: nasopharynx, oropharynx, pharynx,laryngopharynx/hypopharynx, larynx, trachea, bronchi, bronchioles,alveolar ducts and alveoli.

For example, dry powder can be dispensed by an airway delivery devicethat comprises a mechanism for dispensing unit doses, e.g., into achamber from which the drug product is inhaled by a subject and/orexpelled into the subject's airway by any useful mechanism, such as byair flow or pressurized gas. Drug products for certain asthmamedications, such as PULMICORT FLEXHALER and ADVAIR DISKUS utilize suchdelivery devices. Those technologies can readily be applied to thedelivery of the powdered ECM-derived materials described herein. Thepowdered ECM-derived material may be admixed with any pharmaceuticallyacceptable carrier or carriers or other excipients, including flowenhancers, flavorings, salts, buffers, etc.

The powdered or solubilized material also can be dispensed in any usefulspray, aerosol or nebulization airway delivery device. A multitude ofsuch devices are known in the arts. Useful excipients include, withoutlimitation: solvents, such as water and optionally organic solvents,such as an alcohol, buffers, salts, surfactants, flavorings,propellants, such as a fluorocarbon or tetrafluoroethane propellant,rheology modifiers, colorants, preservatives, etc., See, e.g., Troy, D.B., Editor, Remington: The Science and Practice of Pharmacy, 21^(st)Edition, Lippincott Williams & Wilkins (2005), pp. 1000-1017 forexemplary devices and manufacturing methods. The device may deliver thedrug via the nose or mouth or intratracheally, by including in additionto the dispenser, an endotracheal tube, or adapter therefore, so thatthe composition can be delivered into a subject's trachea (see, e.g.,U.S. Pat. No. 6,766,801).

Thus provided is an apparatus comprising an airway delivery devicecomprising a composition comprising a ECM-derived material. Any deviceuseful for delivering a dose of a composition, such as a drug product,to a subject's airway is considered to be an airway delivery device,irrespective of mechanism and physical form of the composition to bedelivered or when delivered. For example, any such device describedherein is considered to be an ‘airway delivery device”. The ECM-derivedmaterial can be a dry powder or dispersed within a solvent, optionallyincluding a propellant, for aerosolization. The airway delivery systemis suitable for delivery of and configured to deliver of one or moreunit doses of the composition to a subject's airway, such as a metereddose aerosol delivery system, as are well known in the pharmaceuticalarts.

By “prevention” or “preventing”, it is not meant absolute prevention ofa stated end-result, such as pulmonary fibrosis, but ascertainablereduction of or lessening of fibrosis in a subject, as compared to asubject not treated in the same manner according to the preventativemethods described herein. This ability of a therapeutic regimen to“prevent” a stated condition typically is ascertained by controlledstudies and is identified by a statistically-significant difference inresults that is medically and/or pharmaceutically acceptable (see, e.g.,FIG. 2).

Any relevant end-point can be used to determine an amount of ECM-derivedmaterial effective to prevent, lessen or treat pulmonary fibrosis in asubject. The amounts effective in a subject in humans or other animalscan be tested in animals, for example as described herein. Effectivedoses can be determined by reference to a therapeutic window between aminimum and maximum dose. The minimum dose is a dosage below which theECM-derived material is not effective in achieving a desired clinicaloutcome. The maximum dosage is a dosage above which the drug causesundesirable and/or unacceptable side-effects, including death or otherside-effects. The maximum dosage also may be limited by the maximumsolubility of the active agent in the drug product composition and theamount of the composition that can be administered at one time. By“effective,” it is meant that pulmonary fibrosis is prevented ormitigated (reduced) to a clinically or statistically-relevant degree ina subject or population of subjects acceptable in the medical,pharmaceutical and/or veterinary arts.

The amount of an ECM-derived material useful in preventing or mitigatingpulmonary fibrosis in a subject will vary, depending on the materialused, and delivery method, among other factors. For example and withoutlimitation, effective unit doses may range from between 10 μg(micrograms) and 1000 mg of the ECM-derived material. In one embodiment,a dry powder of the ECM-derived material is administered by inhalationin a formulation that is suitable for administration by an inhalationroute. In another embodiment, a solution comprising, without limitation,from 1 μg/mL to 100 mg/mL, 100 μg/mL to 10 μg/mL or 1 mg/mL to 5 μg/mL,including 4 mg/mL, or increments within any of the stated ranges, of theECM-derived material is administered to a subject. The material may beadministered in a single unit dose or in multiple doses over anysuitable and effective time period, for example, from one to ten timesdaily, every other day, weekly, bi-weekly, monthly, etc. The quantity ofECM-derived material that is administered to a subject depends on theconcentration of the active agent in the solution, the size of thesubject (a larger subject might better physically tolerate largeramounts of a solution), as well as the maximum tolerable dose and theminimum effective dose, all of which can readily be identified by thoseof ordinary skill in the art. The composition can be administered priorto, during or after exposure of a subject to a fibrosis-inducing event,such as exposure to chemotherapy or radiotherapy in a cancer patient orexposure of a subject to asbestos or a silicious material.

The ECM-derived material may be compounded or otherwise manufacturedinto a suitable composition for use, such as a pharmaceutical dosageform or drug product in which the compound is an active ingredient.Compositions may comprise a pharmaceutically acceptable carrier, orexcipient. An excipient is an inactive substance used as a carrier forthe active ingredients of a medication. Although “inactive,” excipientsmay facilitate and aid in increasing the delivery or bioavailability ofan active ingredient in a drug product. Non-limiting examples of usefulexcipients include: antiadherents, binders, rheology modifiers,coatings, disintegrants, emulsifiers, oils, buffers, salts, acids,bases, fillers, diluents, solvents, flavors, colorants, glidants,lubricants, preservatives, antioxidants, sorbents, vitamins, sweeteners,etc., as are available in the pharmaceutical/compounding arts (see,Troy, D B, Editor, Remington: The Science and Practice of Pharmacy,21^(st) Ed., Lippincott Williams & Wilkins (2005) for detaileddescriptions of various dosage forms, methods of manufacture of suchdosage forms and routes of administration of such dosage forms).Excipients may include compounds or compositions that enhance thesolubility of the compound.

EXAMPLE 1

Regenerative medicine approaches using extracellular matrix (ECM)scaffolds derived from porcine small intestinal submucosa (SIS) andurinary bladder matrix (UBM) have been shown to promote site-specificconstructive tissue remodeling in various body systems (Badylak, S. F.,Biomaterials, 2007. 28(25): p. 3587-93). The site-specific remodelingresponse is due in part to rapid degradation with release of bioactivedegradation products (Gilbert, T. W., et al., J Bone Joint Surg Am,2007. 89(3): p. 621-30 and Reing, J. E., et al., Tissue Eng, 2008. InPress) and to modulation of the immune response to an accommodative,non-cytotoxic response (Badylak, S. F. and Gilbert T. W., Semin Immunol,2008. 20(2): p. 109-16). Based on these findings, the present studytests the hypothesis that UBM-ECM will can promote normal remodeling andprevent fibrosis in the lung using a model of bleomycin-inducedpulmonary fibrosis.

Materials and Methods

Ten-week-old C57BL/6 mice were treated by intratracheal instillationwith 0.1 mL of a solution comprising 0.05 units of bleomycin or salinevehicle with or without 4 mg/mL of porcine UBM-ECM lyophilized powder(particles <75 μM, produced as previously described (Gilbert, T. W., etal., Biomaterials, 2005. 26(12): p. 1431-5)).

The ECM was isolated from the porcine urinary bladder. The bladders wereharvested immediately following euthanasia of market weight pigs(approximately 120 kg). The ECM isolation was performed by removing thetunica muscularis externa and tunica submucosa layers, leaving thebasement membrane and tunica propria intact. The UBM was then washed ina 0.1% peracetic acid solution for 2 h with subsequent rinses inphosphate buffered saline and distilled water to disinfect the materialand remove any cellular remnants. The disinfected material waslyophilized and then chopped into small sheets for immersion in liquidnitrogen. The snap-frozen material was then reduced to small pieces witha Waring blender so that the particles were small enough to be placed ina rotary knife mill. A Wiley mill fitted with a #60 screen was used torestrict the collected powder size to less than 250 mm.

A Sonic Sifter with a series of mesh was utilized to obtain a fractionhaving a particle size of <75 Mm. The smallest mesh size was L3S200, forwhich the opening size is 75 micron. Everything that passed through thelast screen was used.

Mice were sacrificed 14 days after exposure and bronchoalveolar lavagefluid (BALF) was collected by instillation and recovery of 0.8 mL of0.9% saline. Total cell counts, cell differentials, and protein levelsin the BALF were obtained. Lungs were inflation fixed with 10% formalinand hematoxylin and eosin staining was performed on lung sections andscored by a pathologist blinded to the sample groups. All comparisonsbetween groups were compared with one-way ANOVA with Tukey's post-testor two-way ANOVA with Bonferroni's post-test using Graphpad Prism 4. A pvalue of ≦0.05 was considered statistically significant.

Results

To assess inflammation, inflammatory cell accumulation in the BALFpost-treatment was determined. Compared to saline-treated and salinewith UBM-ECM-treated controls, bleomycin-treated mice with and withoutUBM-ECM had more inflammatory cells in their BALF. Furthermore, celldifferentials revealed that there was a significant increase inmacrophages (p<0.001) and modest increases in neutrophils andlymphocytes in both bleomycin and bleomycin with UBM-ECM mice whencompared to both saline controls.

In contrast to inflammation, the amount of fibrotic injury in the lungsfollowing bleomycin was reduced with UBM-ECM treatment. Mice treatedwith bleomycin and UBM-ECM trended to have less protein in their BALFthan mice treated with bleomycin alone. More impressively, histologicalexamination of the lungs of these mice revealed that mice treated withbleomycin and UBM-ECM had significantly less pulmonary fibrosis thanmice treated with only bleomycin (FIG. 2, p<0.001). Finally, treatmentwith UBM-ECM in saline vehicle revealed that UBM-ECM alone did not causeany histological changes in the lung.

Conclusions

Although there was no change in the amount of inflammation and only amodest decrease in protein in the BALF, UBM-ECM was able to dramaticallylimit the fibrosis that results from bleomycin injury. Overall, ourresults strongly suggest that UBM-ECM can minimize bleomycin-inducedpulmonary fibrosis.

EXAMPLE 2 Materials and Methods Preparation of ECM

The preparation of UBM and SIS has been previously described (Brown B,Lindberg K, Reing J, Stolz D B, Badylak S F. The basement membranecomponent of biologic scaffolds derived from extracellular matrix.Tissue Eng 2006; 12(3):519-526). Porcine urinary bladders and smallintestine were harvested from market weight pigs (approximately 110-130kg) immediately after sacrifice at an abattoir and transported to thelab on ice. The urothelial layer of the bladders was removed by soakingthe material in 1 N saline. The tunica serosa, tunica muscularisexterna, tunica submucosa, and most of the muscularis mucosa weremechanically delaminated from the bladder tissue. The remaining basementmembrane of the tunica epithelialis mucosa and the subjacent tunicapropria were collectively termed UBM. For SIS, the tunica muscularisexterna and the majority of the tunica mucosa of the small intestine wasremoved. The remaining tunica submucosa and basilar portion of thetunica mucosa consists of extracellular matrix and the constituentcells, collectively termed SIS. Both UBM and SIS were decellularized anddisinfected by immersion in 0.1% (v/v) peracetic acid (PAA), 4% (v/v)ethanol, and 96% (v/v) deionized water (diH₂O) for 2 h. The material wasthen washed twice for 15 min with PBS (pH=7.4) and twice for 15 min withdiH₂O (12).

After the ECM material was decellularized and disinfected, the scaffoldwas lyophilized and chopped into small sheets. The chopped material wasthen fed through a rotary knife mill. A #60 screen was used to restrictthe collected powder size to less than 250 μm. The powder was thensifted through stainless steel mesh on a Sonic Sifter to less than 75μm. For cell culture and animals studies, the particulate material wasterminally sterilized with 2 MRad γ-irradiation (Gilbert T W, Stolz D B,Biancaniello F, Simmons-Byrd A, Badylak S F. Production andcharacterization of ecm powder: Implications for tissue engineeringapplications. Biomaterials 2005; 26(12):1431-1435). Nonsterileparticulate UBM was added to 1 mg/mL pepsin (Sigma) in 0.01 N HCl for afinal concentration of 10 mg UBMM/mL suspension. The suspension wasmixed on a stir plate at room temperature for approximately 48 hoursuntil no visible pieces of UBM remained. Pepsin buffer control sampleswere prepared by mixing the pepsin digestion buffer (1 mg/mL pepsin in0.01 N HCl) at room temperature for 48 hours.

Animals

All treatments were done intratracheally as previously described(Fattman C L, Chu C T, Kulich S M, Enghild J J, Oury T D. Alteredexpression of extracellular superoxide dismutase in mouse lung afterbleomycin treatment. Free Radic Biol Med 2001; 31(10):1198-1207).Ten-week-old C57BL/6 mice were intratracheally instilled with 0.07 unitsof bleomycin sulfate (Hospira, Inc, Lake Forest, Ill.) with or without280 μg UBM-ECM powder, SIS-ECM powder, UBM-ECM digest, or pepsin bufferdigest control to determine the effect of these ECM compounds on thedevelopment of fibrosis. Control mice were treated with 0.9% salinevehicle with and without 280 μg of ECM. Mice were euthanized 14 daysafter exposure. Bronchoalveolar lavage fluid (BALF) was obtained by theintratracheal instillation and recovery of 0.8 ml of 0.9% saline aspreviously described (Fattman C L, et al., Free Radic Biol Med 2001;31(10):1198-1207). Lungs were inflation fixed with 10% buffered formalinand paraffin embedded for histological analysis.

Bronchoalveolar Lavage Fluid

Total protein was determined by use of the Coomassie Plus Protein AssayReagent (Pierce, Rockford, Ill.). Total white blood cell counts wereobtained with a Beckman Z1 Coulter particle counter (Beckman Coulter,Fullerton, Calif.). To obtain a differential count, bronchoalveolarlavage (BAL) fluid samples were adhered to glass slides with a cytospin,stained with DiffQuik, and the numbers of macrophages, neutrophils,lymphocytes, and eosinophils were counted under a microscope. A total of400 cells were counted per slide. The remaining BAL fluid was spun downat 200×g and supernatants were stored at −70° C. until use for proteinand cytokine analyses.

Histology and Fibrosis Scoring

Standard hematoxylin and eosin staining was performed on 5-μm-thick lungsections as previously described (Fattman C L, et al., Free Radic BiolMed 2001; 31(10):1198-1207). Hematoxylin and eosin-stained sections werescored as previously described (Englert J M, et al. A role for thereceptor for advanced glycation end products in idiopathic pulmonaryfibrosis. Am J Pathol 2008;172(3):583-591 and Fattman C L, et al.Increased sensitivity to asbestos-induced lung injury in mice lackingextracellular superoxide dismutase. Free Radic Biol Med 2006;40(4):601-607) by a pathologist (T.D.O.) who was blinded to samplegroups. Individual fields were examined with a light microscope at ×200magnification. Briefly, every field in the entire lung was scored,starting peripherally. To be counted, each field had to contain alveolartissue in >50% of the field. Scoring in each field was based on thepercentage of alveolar tissue with interstitial fibrosis according tothe following scale: 0=no fibrosis, 1=up to 25%, 2=25-50%, 3=50-75%,4=75-100%. The pathological index score was then reported as a ratio ofthe sum of all of the scores divided by the total number of fieldscounted for each sample. Group scores were averaged for statisticalanalyses.

Toxicity Assay

A549 human epithelial cells (ATCC) were cultured in F 12K mediasupplemented with 10% fetal bovine serum (FBS). A549 cells were platedon 96 well plates (5,000 cells/well). Cells were serum starved for 4 hrsprior to treatment with either 0.02 units of bleomycin, or bleomycinwith various amount of ECM as indicated in serum-free media. 24 hrfollowing treatment, cell viability was measured using CellTiter 96 AQNon-radioactive Assay according to manufacturer's instructions (Promega,Madison, Wis.).

Chemotaxis Assay

Responses of epithelial cells to UBM degradation products werequantitatively evaluated utilizing the Neuro Probe 48-wellmicrochemotaxis chamber (Neuro Probe, Gaithersburg, MD). A549 cells wereserum-starved for 14-17 h prior to experimentation. Based upon pilotstudies to determine the appropriate filter pore size, 5 μmpolycarbonate chemotaxis filters (Neuro Probe, PFB5) were coated equallyon both sides (by immersion) with 0.05 mg/mL rat tail collagen I (BDBiosciences, San Jose, Calif.) and allowed to dry prior to chamberassembly. 27.5 μL of F12K media (Cellgro, 10-025-CV), F12K mediasupplemented with 10% FBS, 0.5 mg/mL UBM digest, 0.1 mg/mL UBM digest,0.5 mg/mL Pepsin digest, and 0.1 mg/mL Pepsin digest was added to thebottom chamber wells. The filter was placed over the bottom chamber, andthe apparatus was assembled according to the manufacturer'sinstructions. Approximately 30,000 cells were then added to each upperchamber well of the apparatus, and the chamber was incubated for 4 h at37° C. in a humidified atmosphere in 95% air: 5% CO₂. Cells remaining onthe topside of the membrane (i.e., nonmigrated cells) were removed, andthen cells on the bottom side of the membrane (i.e., migrated cells)were stained with Diff Quik (Dade AG, Liederbach, Germany). The filterwas then mounted with Vectashield containing DAPI (Vector Laboratories,H-1200) and fluorescent images of each conditioned well were captured at10× magnification using a Nuance multispectral imaging system and Nikonmicroscope. Each experimental condition was tested in quadruplicate inthree independent experiments. The average number of migrated cells ineach experiment was normalized to the positive control (10% FBS) bycalculating the average percentage of migrated cells as a percentage ofthe positive control for each condition.

Wound Healing Assay

A549 cells cultured in F12K media (Cellgro 10-025-CV) supplemented with10% FBS were seeded and grown to confluence in a 6-well plate. Cellswere serum starved in non-supplemented F12K media overnight prior toinitiation of the experiment. Straight wounds were created as previouslydescribed (Liang C C, et al. In vitro scratch assay: A convenient andinexpensive method for analysis of cell migration in vitro. Nat Protoc2007; 2(2):329-333) by scratching vertically with a p-200 micropipettetip. The wounds were then washed and treated with the followingexperimental conditions: 0.075 U/mL Bleomycin; 0.075 U/mL Bleomycin+0.5mg/mL UBM powder; 0.075 U/mL Bleomycin+0.5 mg/mL UBM digest; 0.075 U/mLBleomycin+0.5 mg/mL Pepsin digest; and 0.5 mg/mL UBM digest. Images werecaptured at 0 and 24 hours after addition of treatments. Using ImageJ,average wound widths in pixels were obtained for each well at each timepoint.

Statistical Analyses

The significance of all quantitative data was assessed using either aStudents t-test or one-way ANOVA with Tukey's post-test (for comparisonof 3 or more groups). Data was analyzed using the computer program Prism(GraphPad Software, San Diego, Calif.). A p-value of less than or equalto 0.05 was considered statistically significant.

Results UBM-ECM Prevents Bleomycin-Induced Pulmonary Fibrosis

Histologic analyses of the lungs showed a significant reduction infibrosis in the lungs of bleomycin-treated mice that received UBM-ECM(FIG. 3C) or UBM-ECM digest (FIG. 3E) when compared to the mice thatreceived bleomycin alone (FIG. 3B) or bleomycin and pepsin digestcontrol (FIG. 3D) respectively. In contrast, bleomycin treated mice thatreceived SIS-ECM (FIG. 3F) showed fibrosis that was not significantlydifferent from bleomycin alone. These differences in fibrosis werequantified by histologic scoring (FIG. 3G). Administration of ECM alonedid not cause any adverse effects and the lungs were pathologicallyidentical to the saline controls (data not shown).

There was an increase in the number of leukocytes within the BAL fluidfollowing bleomycin exposure regardless of ECM treatment (FIG. 4A-C).The BAL fluid differentials showed a significant increase in macrophagesand modest increases in neutrophils and lymphocytes in the BALF of micetreated with bleomycin and bleomycin with various ECM treatments whencompared to controls (FIG. 4D-F).

UBM-ECM Does Not Affect Bleomycin Toxicity

A549 cells were treated with bleomycin and different doses of UBM-ECM,SIS-ECM, or ECM digest to verify that the presence of the ECM scaffoldmaterial does not alter the toxicity of the bleomycin. These resultsshowed that reduction in cell viability caused by bleomycin was notsignificantly altered due to bleomycin treatment simultaneous withUBM-ECM (FIG. 5A), SIS-ECM (FIG. 5B), or UBM-ECM Digest (FIG. 5C).

UBM-ECM Promotes Migration of Epithelial Cells

Migration of serum-starved A549 cells towards the UBM-ECM digestapproached the migration promoted by the positive control, 10% FBS (FIG.6). The UBM digest at 500 μg/ml concentration showed 94%±35% of the cellmigration observed for the positive control, while 100 μg/mlconcentration showed 73%±20% of the cell migration observed for thepositive control. The relative cell migration towards the pepsin digestcontrols were 23%±17% and 19%±18% for 500 μg/ml and 100 μg/mlconcentration respectively. The migration toward both concentrations ofpepsin digest was similar to the F12K media alone (13%±11%). At bothconcentrations, the UBM-ECM digest showed significantly improvedmigration compared to the pepsin digest control (FIG. 6).

Using a wound healing assay, serum-starved A549 cells were treated withUBM-ECM or UBM-ECM digest in the presence of bleomycin to evaluate theability of ECM to improve wound repair. UBM-ECM digest treatmentpromoted wound closure in the presence of bleomycin while pepsin digestcontrol did not affect wound width (FIG. 7). UBM-ECM treatment showed atrend toward improved wound closure, but did not significantly reducewound width in the presence of bleomycin.

UBM-ECM scaffold material prevented bleomycin-induced pulmonary fibrosiswhen delivered simultaneously with the bleomycin regardless of the form(particulate or digested) of the material. UBM-ECM treatmentsignificantly reduced the histologic presentation of fibrosis, such thatthere was no significant difference between the histologic appearance ofthe lungs in animals treated with bleomycin and UBM-ECM as compared tothose treated with the saline alone. In contrast, simultaneous treatmentof bleomycin exposed animals with SIS-ECM showed a significant increasein fibrosis as compared to saline alone. The SIS-ECM treated grouptended to have less fibrosis than bleomycin alone, but was notsignificantly different. It is clear that the attenuation of fibrosis inresponse to UBM-ECM was not due to neutralization of the bleomycin ascell culture studies showed that UBM-ECM did not prevent cell death.Furthermore, animals treated with bleomycin and ECM products had thesame increase in the number of inflammatory cells and similar cellularcomposition in the BAL fluid as animals treated with bleomycin alone.These results suggest that UBM-ECM limited bleomycin-inducedfibrogenesis and did not do so by interfering with the effects ofbleomycin.

Epithelial cell damage and cell death causes gaps in the epithelialbasement membrane allowing for fibroblast migration, which then promotefibrosis. The different responses observed in response to UBM-ECM andSIS-ECM suggests a role for the basement membrane in the reparativeprocess. UBM-ECM is known to possess an intact basement membrane, whileSIS-ECM lacks the basement membrane due to the specific tissue layersfrom which each is isolated (Brown B, Lindberg K, Reing J, Stolz D B,Badylak S F. The basement membrane component of biologic scaffoldsderived from extracellular matrix. Tissue Eng 2006; 12(3):519-526). Thepresence of a basement membrane structure has been shown to promotehealing in various organs, including the lungs, by providing guidancefor re-epithelialization and separating the epithelium from theinterstitial connective tissue (Vracko R. Basal lamina scaffold-anatomyand significance for maintenance of orderly tissue structure. Am JPathol 1974; 77(2):314-346). This is consistent with the findings ofprevious studies in which UBM-ECM promoted the formation of apseudostratified, columnar, ciliated epithelium when used for patchtracheoplasty in a dog model (Gilbert T W, Gilbert S, Madden M, ReynoldsS D, Badylak S F. Morphologic assessment of extracellular matrixscaffolds for patch tracheoplasty in a canine model. Ann Thorac Surg2008; 86(3):967-973; discussion 973-974). Furthermore, chemotaxisexperiments in the present study showed that the digested form ofUBM-ECM promoted migration of airway epithelial cells. These fmdingssuggest that the presence of UBM-ECM promotes re-epithelialization atthe site of airway epithelial injury caused by bleomycin.

Furthermore, the findings suggest that degradation of the ECM is animportant component of the host response. Previous studies have shownthat ECM degradation products recruit progenitor cells to the site ofremodeling (Beattie A J, et al. Chemoattraction of progenitor cells byremodeling extracellular matrix scaffolds. Tissue Eng 2008; doi:10.1089/ten.tea.2008.0162; Brennan E P, et al. J Tissue Eng Regen Med2008; 2(8):491-498; and Reing J E, et al. Degradation products ofextracellular matrix affect cell migration and proliferation. Tissue EngPart A 2009; 15(3):605-614), promote angiogenesis (Li F, et al.Low-molecular-weight peptides derived from extracellular matrix aschemoattractants for primary endothelial cells. Endothelium 2004;11(3-4):199-206), and provide bacteriostasis (Brennan E P, et al.Antibacterial activity within degradation products of biologicalscaffolds composed of extracellular matrix. Tissue Eng 2006;12(10):2949-2955). In vivo studies have shown that ECM scaffolds degradewithin 3 months after implantation to repair load bearing tissues(Gilbert T W, et al. Degradation and remodeling of small intestinalsubmucosa in canine achilles tendon repair. J Bone Joint Surg Am 2007;89(3):621-630 and Record R D, et al. In vivo degradation of ¹⁴c-labeledsmall intestinal submucosa (sis) when used for urinary bladder repair.Biomaterials 2001; 22(19):2653-2659). The kinetics of ECM degradationafter instillation into an injured lung is unknown and would requireadditional study.

Based on previous studies, it is known that oxidative fragmentation ofECM components such as heparin sulfate and collagen influence thedevelopment of fibrosis (Gao F, et al. Extracellular superoxidedismutase inhibits inflammation by preventing oxidative fragmentation ofhyaluronan. J Biol Chem 2008; 283(10):6058-6066; Kliment C R, et al.Oxidative stress alters syndecan-1 distribution in lungs with pulmonaryfibrosis. J Biol Chem 2009; 284(6):3537-3545; and Kliment C R, et al.Extracellular superoxide dismutase protects against matrix degradationof heparan sulfate in the lung. Antioxid Redox Signal 2008;10(2):261-268). However, the present study showed that intactparticulate UBM-ECM and enzymatically digested UBM-ECM preventedfibrosis. It is known that ECM scaffold material is a mixture of ECMproteins and other substances, such as growth factors (Badylak S F, etal. Extracellular matrix as a biological scaffold material: Structureand function. Acta Biomater 2009; 5(1):1-13). It is likely that thiscomplex composition of ECM promotes healing that outweighs thedetrimental effects of the proteoglycans and glycosoaminoglycansdegradation products in these ECM materials.

In conclusion, this study shows that basement membrane containing ECMprotected against pulmonary fibrosis using a bleomycin model. Thesefindings also strongly suggest that one mechanism in which this ECMmaterial prevents fibrosis is by promoting epithelial cell chemotaxisand re-epithelialization. These results support the conclusion that ECMwill have therapeutic benefit if administered after injury has begun.These findings also suggest that ECM would be useful in attenuation ofthe response to chronic environmental irritants, such as asbestos andsilica, and in different forms of pulmonary fibrosis, and in modulationof immune response.

EXAMPLE 3

Chemotherapy and radiotherapy patients often develop pulmonary fibrosis.Drugs such as bleomycin, cyclophosphamide, busulfan, chlorambucil,oxaliplatin, 5-fluorouracil, and nitrosourea drugs are known to causepulmonary fibrosis. Delivery of the ECM powder could also be used toprevent pulmonary fibrosis in patients being treated with radiationtherapy. Therefore, in an extension of the work described above, C57BL/6mice were intratracheally instilled with 0.07 units of bleomycin sulfate(Hospira, Inc, Lake Forest, Ill.) or 0.9% saline as a control (total of70 μL/mouse). Seven days after administration of bleomycin sulfate orsaline, mice were intratracheally treated with 280 μg ECM-derivedmaterial described above in Example 1 or 0.9% saline control (total of70 μL/mouse) to see if ECM-derived material could prevent furtherdevelopment of fibrosis as shown in FIG. 8. This further illustratesthat ECM-derived material is potentially useful as a treatment orpre-treatment for patients undergoing chemotherapy and radiotherapy.

Having described this invention, it will be understood to those ofordinary skill in the art that the same can be performed within a wideand equivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any embodiment thereof.

We claim:
 1. An apparatus for reducing or preventing development ofinterstitial lung disease in a subject, comprising: an airway deliverysystem to administer via the subject's airway a composition comprising adevitalized, decellularized extracellular matrix material comprisingepithelial basement membrane that is delaminated from one or more layersof a devitalized epithelial tissue in an amount that is effective toreduce or prevent development interstitial lung disease in the subject,said airway delivery system selected from the group consisting of ameter-dosed inhaler, nebulizer, spray, and aerosolizer.
 2. The apparatusof claim 1, wherein the airway delivery system is a metered doseinhaler.
 3. The apparatus of claim 1, wherein the airway delivery systemis a metered-dose device and the composition further comprises apropellant.
 4. The apparatus of claim 1, wherein the compositioncomprises a dry powder.
 5. The apparatus of claim 1, wherein theapparatus is an intra-tracheal delivery device.
 6. The apparatus ofclaim 1, wherein the airway delivery system comprises an endotrachealtube or an adapter for an endotracheal tube.
 7. The apparatus of claim1, wherein the composition comprises a liquid.
 8. The apparatus of claim4, wherein the powdered composition has a maximum particle size of 250μM.
 9. The apparatus of claim 4, wherein the powdered composition has amaximum particle size of 75 μM.
 10. The apparatus of claim 1, whereinthe extracellular matrix material is derived from urinary bladder. 11.The apparatus of claim 1, wherein the interstitial lung disease isassociated with silicosis; asbestosis; berylliosis; hypersensitivitypneumonitis; drug induced interstitial lung disease, connective tissuedisease, systemic sclerosis, dermatomyositis, systemic lupuserythematosus, rheumatoid arthritis; infection, atypical pneumonia,pneumocystis pneumonia (PCP), tuberculosis; idiopathic interstitial lungdisease, sarcoidosis, idiopathic pulmonary fibrosis, Hamman-Richsyndrome, a malignancy, or lymphangitic carcinomatosis.
 12. Theapparatus of claim 1, wherein said airway delivery system is capable ofdelivering of about 10 μg to about 1000 mg of the composition to thesubject.
 13. The apparatus of claim 1, wherein said airway deliverysystem is capable of delivery an amount of said composition in asolution to the subject in the range of 1 μg/ml to 100 mg/ml, 100 μg/mlto 10 μg/ml, or 5 μg/ml to 1 mg/ml, or 4 mg/ml.
 14. The apparatus ofclaim 1, wherein said extracellular matrix material further comprisestunica propria.
 15. The apparatus of claim 14, wherein the extracellularmatrix material further comprises submucosa.
 16. The apparatus of claim1, wherein the composition is solubilized.
 17. The apparatus of claim 1,wherein said extracellular matrix material is derived from a tissueselected from the group consisting of trachea, lung, small intestine,and skin.
 18. A compound for reducing or preventing interstitial lungdisease in a patient, comprising: an ECM-derived material comprisingepithelial basement membrane; and an excipient selected from the groupconsisting of antiadherents, binders, rheology modifiers, coatings,disintegrants, emulsifiers, oils, buffers, salts, acids, bases, fillers,diluents, solvents, flavors, colorants, glidants, lubricants,preservatives, antioxidants, sorbents, vitamins, sweeteners andcompositions that enhance solubility of the compound.